Bottom Line:
We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments.Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system.Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells.

Affiliation: Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACTWe have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

Figure 8: (A and B) HRP distribution in cells expressing ARF6(Q67L) and ARF6(T27N). Cells expressing ARF6(Q67L) (A) and ARF6(T27N) (B) were incubated with HRP for 60 min, fixed, and then processed for ultrathin cryosections. Sections were first labeled with anti-ARF6 antibody followed by protein A–conjugated gold particles (10 nm) and then with an anti-HRP antibody followed by protein A–gold particles (5 nm). (A) Typically, in ARF6 (Q67L)-expressing cells, HRP labeling was restricted to small vesicles at the periphery (e). Membrane invaginations were observed even at very low levels of ARF6(Q67L) expression, implicit by low numbers of gold particles on the membrane. (B) A small population of ARF6(T27N)-positive vesicles were also labeled with HRP (arrows). i, invaginations; e, endosomes; p, plasma membrane. Bar, 200 nm.

Mentions:
To determine the subcellular distribution of ARF6 relative to HRP under conditions of both short and more prolonged exposure to the tracer, cells expressing either wild-type ARF6 or its mutants defective in either GTP binding or hydrolysis were incubated with HRP for 5 or 60 min, fixed, processed for cryoimmunoelectron microscopy, and then labeled with antibodies directed against ARF6 and HRP. In cells expressing wild-type ARF6, no colocalization between these markers was observed with 5 min of HRP uptake (Table II), and ARF6-positive vesicles were clearly distinct from the numerous early endocytic structures that labeled with HRP. This strongly suggests that ARF6-positive vesicles were not early endosomes. On more prolonged exposure to HRP however, <10% of ARF6-positive vesicles were also labeled with HRP (Fig. 7). In cells expressing ARF6(Q67L), the GTPase-defective mutant, HRP label appeared to be significantly lower and exhibited <50% of gold labeling compared with control cells, thus indicating that the amount of HRP internalized into these cells was significantly reduced. This is similar to what was observed previously for the internalization of transferrin in CHO cells on expression of ARF6(Q67L; D'Souza-Schorey et al., 1995). The reduced efficiency of endocytosis in these cells may most likely be due to the dramatic alterations at the plasma membrane induced on expression of the GTPase-defective mutant. Labeling for HRP was restricted to small 80–100-nm vesicles at the periphery (Fig. 8 A), and on occasion, some label was also seen “entrapped” in membrane invaginations, but in 95% of these cells the ARF6-positive plasma membrane structures were devoid of HRP labeling. In cells expressing the GTP binding–defective mutant, ARF6(T27N), similar to what was observed with wild-type ARF6, no colocalization between the ARF6-positive and HRP-positive vesicles was observed with a short 5-min exposure to HRP (see Table II). However, after 60 min of HRP uptake, ∼10–15% of ARF6-positive vesicles that accumulate at the pericentriolar region of the cell also labeled for HRP (Fig. 8 B). Vesicles that label for ARF6(T27N), but not HRP, may be preformed vesicles or vesicles that are not accessible to HRP. These findings indicate that at some step after internalization, most likely at the recycling endosome, a small proportion of HRP has access to ARF6-positive vesicles. Based on the these observations and the findings described above on the colocalization of ARF6 with Tfn-Rs and cellubrevin, we propose that ARF6 localizes to and regulates the transport of recycling vesicles at the pericentriolar region of CHO cells to the plasma membrane.

Figure 8: (A and B) HRP distribution in cells expressing ARF6(Q67L) and ARF6(T27N). Cells expressing ARF6(Q67L) (A) and ARF6(T27N) (B) were incubated with HRP for 60 min, fixed, and then processed for ultrathin cryosections. Sections were first labeled with anti-ARF6 antibody followed by protein A–conjugated gold particles (10 nm) and then with an anti-HRP antibody followed by protein A–gold particles (5 nm). (A) Typically, in ARF6 (Q67L)-expressing cells, HRP labeling was restricted to small vesicles at the periphery (e). Membrane invaginations were observed even at very low levels of ARF6(Q67L) expression, implicit by low numbers of gold particles on the membrane. (B) A small population of ARF6(T27N)-positive vesicles were also labeled with HRP (arrows). i, invaginations; e, endosomes; p, plasma membrane. Bar, 200 nm.

Mentions:
To determine the subcellular distribution of ARF6 relative to HRP under conditions of both short and more prolonged exposure to the tracer, cells expressing either wild-type ARF6 or its mutants defective in either GTP binding or hydrolysis were incubated with HRP for 5 or 60 min, fixed, processed for cryoimmunoelectron microscopy, and then labeled with antibodies directed against ARF6 and HRP. In cells expressing wild-type ARF6, no colocalization between these markers was observed with 5 min of HRP uptake (Table II), and ARF6-positive vesicles were clearly distinct from the numerous early endocytic structures that labeled with HRP. This strongly suggests that ARF6-positive vesicles were not early endosomes. On more prolonged exposure to HRP however, <10% of ARF6-positive vesicles were also labeled with HRP (Fig. 7). In cells expressing ARF6(Q67L), the GTPase-defective mutant, HRP label appeared to be significantly lower and exhibited <50% of gold labeling compared with control cells, thus indicating that the amount of HRP internalized into these cells was significantly reduced. This is similar to what was observed previously for the internalization of transferrin in CHO cells on expression of ARF6(Q67L; D'Souza-Schorey et al., 1995). The reduced efficiency of endocytosis in these cells may most likely be due to the dramatic alterations at the plasma membrane induced on expression of the GTPase-defective mutant. Labeling for HRP was restricted to small 80–100-nm vesicles at the periphery (Fig. 8 A), and on occasion, some label was also seen “entrapped” in membrane invaginations, but in 95% of these cells the ARF6-positive plasma membrane structures were devoid of HRP labeling. In cells expressing the GTP binding–defective mutant, ARF6(T27N), similar to what was observed with wild-type ARF6, no colocalization between the ARF6-positive and HRP-positive vesicles was observed with a short 5-min exposure to HRP (see Table II). However, after 60 min of HRP uptake, ∼10–15% of ARF6-positive vesicles that accumulate at the pericentriolar region of the cell also labeled for HRP (Fig. 8 B). Vesicles that label for ARF6(T27N), but not HRP, may be preformed vesicles or vesicles that are not accessible to HRP. These findings indicate that at some step after internalization, most likely at the recycling endosome, a small proportion of HRP has access to ARF6-positive vesicles. Based on the these observations and the findings described above on the colocalization of ARF6 with Tfn-Rs and cellubrevin, we propose that ARF6 localizes to and regulates the transport of recycling vesicles at the pericentriolar region of CHO cells to the plasma membrane.

Bottom Line:
We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments.Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system.Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells.

Affiliation:
Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACTWe have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.